Cancer nanomedicine: a review of nano-therapeutics and challenges ahead

Cancer is known as the most dangerous disease in the world in terms of mortality and lack of effective treatment. Research on cancer treatment is still active and of great social importance. Since 1930, chemotherapeutics have been used to treat cancer. However, such conventional treatments are associated with pain, side effects, and a lack of targeting. Nanomedicines are an emerging alternative due to their targeting, bioavailability, and low toxicity. Nanoparticles target cancer cells via active and passive mechanisms. Since FDA approval for Doxil®, several nano-therapeutics have been developed, and a few have received approval for use in cancer treatment. Along with liposomes, solid lipid nanoparticles, polymeric nanoparticles, and nanoemulsions, even newer techniques involving extracellular vesicles (EVs) and thermal nanomaterials are now being researched and implemented in practice. This review highlights the evolution and current status of cancer therapy, with a focus on clinical/pre-clinical nanomedicine cancer studies. Insight is also provided into the prospects in this regard.

metabolic regulation as well as the ability to spread to other parts of the body (metastasis). 4 These hallmarks of cancer alter the microenvironment of the tumor site, and actively route the energy and nutrient supply for its growth, rather than for bodily functions. 5 This also results in heterogeneity and mixed populations of cells, each of which has a different response to various therapeutic approaches. 6 Many therapeutic methods have been tested against cancer with varying levels of success, 7 the most common ones being chemotherapy, radiotherapy, and surgery. 8 Chemotherapy started in the early 1930s with the use of chemicals against tumor cells, prominently with the use of nitrogen mustard against leukemia, 9 and alkylating drugs such aschlorambucil. 10 However, the cure for cancer is still evasive, probably owing to the diverse population of cells present in the tumor environment, and the inability of the above-mentioned methods to effectively regulate the tumor microenvironment and the various hallmarks of cancer. 11 Nowadays, different types of cancer are observed among the population, indicating a high death rate and increasing live cases among children. The mortality rate of several cancers and extrapolated future projections are shown in Fig. 1. According to World Health Organization (WHO), there were 10 million deaths attributable to cancer in 2020, and each year approximately 400 000 children develop cancer. It is expected that by 2040, new cases will rise to 29.5 million with 16.4 million cancer-related deaths. 12 Nano-oncology is an emerging strategy in cancer therapy that involves the use of nano-dimensional therapeutic materials as anticancer agents, and it has generated promising results in research and clinical trials. 13 This is a eld that has grown tremendously in the past two decades with the development of new approaches not only for cancer treatment but also for diagnosis and prevention. 14 Nanotechnology has become an innovative method to treat various diseases, owing to its high potential and treatment efficacy in different cancer types. Cancer nanomedicine has wide applications in effective tumor therapy, based on targeting, imaging, viral nanoparticles, and enhanced delivery. 15 In this review, we present the current status of the research in nano-oncology. Also, a review of promising nano-oncological Dr Athira Johnson is currently working as Postdoctoral fellow at the Indian Institute of Technology Madras, Tamil Nadu, India. She received both her Bachelors and Master's degree from Mahatma Gandhi University, Kottayam, Kerala, India, and her PhD from National Taiwan Ocean University, Keelung, Taiwan in the eld of Nanomedicine. Her current research topic includes formulation, characterization and in vitro drug release of drug delivery system for cancer therapy. products in various stages of clinical and preclinical trials is provided.

Origin and history of cancer nanomedicine
The early history of nanomedicine may be dated back to ancient times when colloidal gold particles were used for medicinal purposes. 16 Ancient medical literature has a record of drug preparation methods that use pulverization of medicines to obtain required consistencies, a process later shown to be a nano-scale preparation technique. 17 Nanomedicine in its current form has been considered a possibility ever since the concept of nanotechnology was rst introduced in 1959 by Richard Feynman in his Caltech talk, "There is plenty of room at the bottom". He mentioned that it would be possible to arrange the atoms as desired. Nanomedicine can be dened as nanotechnology, which deals with the size range of 1 to 100 nm, applied to health and medicine. 18 In 1999, Robert A Freitas introduced the term "nanomedicine", and it has been used extensively in technical literature since then. Nanomedicine has been sought due to the deciencies that exist in treating cancer with drugs of low specicity, rapid drug clearance, biodegradation, and limited targeting. 19 Nanocarriers were proposed as a superior alternative with targeted drug delivery to the tumor tissue and controlled release of the intended drug. 20 Nanoparticles or nanomaterials which are innovated and modied are shown over a timeline in Fig. 2. Cancer therapy based on nanoparticles began with the development of doxorubicinloaded liposomes for treating breast cancer. Later, polymers and dendrimers came into use. Between 2000 to 2015, siRNA molecules with different nanoparticles and solid lipid nanoparticles were developed for targeted therapy and treatment efficacy, respectively. 21 Then, gold nanoparticles and quantum dots were deployed in cancer therapy, especially for bio-imaging. In the future, it is expected that better treatment can be achieved by using Nanobots. 22,23 Nanotechnology has enabled applications in a wide range of elds such as Chemistry, Biology, Physics, Engineering, and Medicine. In the clinical industry, it has facilitated the development of delivery systems of drugs for complicated diseases. [24][25][26] Nanomedicine is a pioneer eld of nanotechnology that deals with the development of powerful techniques for treating diseases and for delivering certain biological compounds for treatment. An injection is the only method of administration for biologics such as peptides, therapeutic proteins, and antibodies (with a few exceptions). Nano-drug delivery has intensied efforts for the development of painless injections, targeted treatment, and an increased ability of the drug to penetrate the BBB (Blood Brain Barrier). 27 In this century, researchers have advanced the uses of nanotechnology in medicine, with the usage of liposome-mediated drug delivery options, and the encapsulation of drugs such as Doxorubicin and Cabilivi™ as nano-sized drugs. Lipid nanoparticles show promising results in mediating synthetic compounds into nano-drugs. 28 Loading mRNA and therapeutic proteins as a nano-drug enhances the clinical applications of nanomedicine. Recently, the application of nanomedicine in immunomodulation and immunotherapy has enabled the use of nanoparticles such as acid-base nano-constructs, extracellular vesicles, and virus-modied exosomes. 29 There are safety concerns with attempts to use nano-medical research in clinical settings, necessitating a better understanding of the crucial characteristics that inuence how nanomaterials interact with tissues and organs. 30 When compared to conventional anticancer therapies, targeted nano-therapy has proved to be a more effective approach with minimum toxicity, enhanced permeability, and retention. It increases the plasma half-life of the nano-size drugs and alters biodistribution, resulting in differential accumulation of nanoparticles in the tumor tissues. 31 The increased plasma halflife of nanoparticles happens when their size exceeds the limit of the renal excretion threshold and limits their clearance. The differential accumulation of nanoparticles in tumor tissues results in a higher concentration of the nano-sized drug in these tumor tissues than in the plasma or other organs; the accumulation is time-dependent and can be reproduced in tumors of different sizes. 32 This phenomenon results in prolonged therapeutic effects in addition to targeting when the pharmacological effects and plasma concentration are synergized. 33 Interest in the application of nanotechnology has increased in cancer therapy because of its advantages in drug delivery, diagnosis, and imaging. 34 Several therapeutic nanoparticle platforms, such as albumin NPs, liposomes, and polymeric micelles, have been approved for cancer treatment. 35 Each has its specic characteristics which make them advantageous for certain uses even while posing limitations in certain others. 36,37 3. Mechanism of targeting by nano drug vehicles A very important criterion for the selection of a nanomedicine formulation for cancer therapy would be its efficiency in targeting the cancer tissue in a specic manner and having minimal side effects on the normal tissue. The various nanoformulations used to deliver anticancer drugs to tumor sites use varying targeting mechanisms for this purpose. The mechanism of drug delivery and the advantages of nanocarriers will vary by carrier. Nanocarriers directly deliver therapeutic agents to the bloodstream and reach the targeted area. They then induce DNA damage by reactive oxygen species (ROS) overproduction. This may nally lead to apoptosis and cell death. 38,39 Two major types of targeting methods are used for nanobased drug delivery: passive and active. In passive method (Fig. 3), the properties of the tumor site are used to concentrate the nano-vehicles to the tumor site. The major factors used for this are Enhanced Permeability and Retention (EPR) and Tumor Micro Environment (TME) properties. Unlike normal cells, tumor cells induce neovascularisation due to high proliferation and large pores in the vascular walls that favor passive targeting. 40 Due to imperfect angiogenesis, particles can reach the tumor site and accumulate. Poor lymphatic drainage also increases particle retention resulting in EPR on tumors. 41 However, the high interstitial uid pressure inside the tumor microenvironment reduces the uptake and homogeneous distribution of nanoparticles. 42 Although the enhanced permeability and retention effect of tumor tissue causes nanoparticles to preferentially accumulate there to a greater extent than in normal tissue, 43 the abnormal and dysfunctional tumor microenvironment frequently leads to the heterogeneous distribution of nanoparticles 44 which primarily reside in the perivascular area and tumor periphery. 45 Therefore, many nanocarriers also utilize the TME properties such as acidic pH, higher redox potential, and differential secretion of lytic enzymes for uniform drug delivery throughout the tumor. Active targeting (Fig. 4) also utilizes the properties of the tumor cells such as the cell surface receptors expressed by the cancer cells. However, the targeting is achieved by the use of various molecules hybridized along with the carrier to specically target these. Here, we look into the different modes of targeting used by the various nano-formulations and some of their advantages as well as disadvantages.
In general, passive targeting is based on the diffusion mechanism and it is affected by various factors such as size, shape, and surface properties. It is noted that high bioavailability and reduced renal clearance can be achieved with 40 to 400 nm by increasing the circulation time. Likewise, maintaining the particle size between 50 to 200 and with a rigid and spherical appearance improves the circulation time and also reduces kidney clearance. 46 Tumor cells were characterized by irregular neovascularization, higher expression of inammatory factors, and lack of an efficient lymphatic drainage system. 47 Because of the leaky vasculature structure and poor lymphatic drainage, nanoparticles will eventually enter into tumor tissues and retain them in the tumor bed due to long circulation time. In tumor cells, EPR effect helps to improve drug accumulation, however, in normal tissues, nanoparticles will be cleared by the mononuclear phagocyte system (MPS) or by glomerular ltration of the kidney. Certain barriers such as abnormal tumor vasculature, growth-induced solid stress, and solid stress from the abnormal stromal matrix will hinder the delivery of nanosized drugs. 48 Gradually, acidic and hypoxic conditions in the tumor cells, heterogeneous perfusion, and elevated interstitial uid pressure prevent nanoparticle penetration. 49 These issues can be solved by improving drug delivery by taking the advantage of EPR effect and TME properties.
Maintaining the size of the nanoparticle is better to improve the EPR effect. In addition, the neutral or negative charge of the particles increases the circulation time and drug accumulation by improving plasma half-lives. Applying some adjuvants like nitric oxide donors to improve the EPR effects also can be done. 47 EPR effect is affected by certain factors such as extravasation, diffusion, and convection in the interstitium, tumor vasculature, and biology, tumor extravascular environment, and physiochemical factors. Also, EPR is heterogenous, in terms of tumor blood ow, hypoxic areas, vascular permeability, extravasation, and penetration. 46 When the nanoparticles enter the body, it passes through different stages including circulation, endocytosis, accumulation, etc. Besides, these particles are highly prone to the opsonization process and because of it; a protein corona will form over the nanoparticles depending on the nanoparticle's characteristics. To avoid this, hydrophilic polymers (PEGylation) with stealth properties can be used to reduce the absorption of opsonins to the nanoparticles. Another strategy is the silencing or depleting of Kupffer cells to escape from the RES system. These cells are specialized macrophages that facilitate the uptake of foreign materials. 50 Recently, a reduction in tumor hypoxia and modulation of polyethyleneimine cytotoxicity by using PEGylated Prussian blue  nanoparticles were reported. It is a dual-enhanced photodynamic therapy with an oxygen self-supply property. Besides, better therapeutic efficacy was observed in breast cancer cells and tumor-bearing mice aer laser irradiation. 51 In another study, evident cell apoptosis and necrosis were observed in PEGylated nanographene oxide-treated cancer cells and this study is an example of combination therapy. 52 A recently published paper examined an iron-dependent regulated cell death process (ferroptosis) by liposomes embedded with PEG-coated 3 nm g-Fe 2 O 3 nanoparticles in the bilayer. These particles promote hydroxyl radical generation and cause efficient lipid peroxidation. The addition of doxorubicin improves the chemotherapeutic effect and traceable magnetic resonance imaging and pH/ROS dual-responsive drug delivery were also visible during the study. 53 In detail, tumor development is affected by genetic/ epigenetic and tumor microenvironment changes (TME). TME consists of tumor cells, stromal broblasts, endothelial cells, immune cells, and extra cellular matrix. Disruption of the interaction between stromal cells and tumors is one of the ways to combat tumor progression. Several strategies such as carcinoma-associated broblast (CAF) depletion, ECM targeting, anti-angiogenic therapy, exosome/circulating tumor cells (CTCs) targeting, and immune modulation/reprogramming can be done for this purpose. 54 Designing a pH-responsive drug delivery system is also one of the ways to target tumor cells. Because of the Warburg effect, a large amount of lactic acid will release and it will help the cells to grow in low oxygen and acidic environment. Designing a pH-responsive nanoparticle, which is stable at physiological pH and degradable at tumor pH is the right track to target tumor cells. 55 Along with this, light-sensitive drug delivery system design is also applicable to cancer treatment. Over expression of matrix metalloproteinase (MMP) is seen in most cancer cells and it can be used as a tumor-specic trigger to tune the size of nanomedicines to enhance tumor penetration. Another possible way is degrading the dense ECM to enhance drug penetration. 56 Recently, researchers developed gelatin/nanochitosan/doxorubicin nanoparticles for cancer therapy. While reaching the tumor site, MMP-2 degrades gelatin from 178 nm GND to release smaller 4 nm nanochitosan/ doxorubicin (ND) nanoparticles for deep tumor penetration and efficient tumor cell endocytosis. Finally, doxorubicin is released due to low pH and MMP-2 activity. In this study, the biocompatibility of the drug was also reported in a mouse tumor bearing model. 57 An aptamer-decorated hypoxiaresponsive nanoparticles (DGL)n@Apt co-loading with gemcitabine monophosphate and STAT3 inhibitor HJC0152 was developed to evaluate its tumor penetration in pancreatic ductal adenocarcinoma cells. This particle can reverse its charge in TME and reduce the size triggered by hypoxia. 58 Immunotherapy is also considered as a promising technology to treat cancer. This technique involves the modulation of cell-specic immune responses toward the tumor. TME contains immunosuppressive cells like tumor associated macrophages (TAMs). So, cancer therapy has been improved by targeting TAMs. 59 Solid tumors contain up to 50% of macrophages and it produces cytokines while recruiting to tumor site by the microenvironment. This process may depend on lactic acid level, inammation, and local anoxia. 60 M1 (proin-ammatory and antitumor) and M2 (anti-inammatory and pro-tumor) are the two subsets of TAMs 61 It is noted that the interaction of immune cells with nanoparticles activates the immune system and thereby can improve therapeutic effects. Tumor growth can be reduced by depolarizing the M2 type to the M1 type and this TAM can serve as a drug depot for the accumulation of nanoparticles. So that, local delivery is also achievable. 62 Recently, the anti-tumor activity of polyethylene glycol-conjugated gold nanoparticles was reported. In this study, nanoparticles suppressed TAMs M2 polarization via induction of lysosome dysfunction and autophagic ux inhibition in both in vitro and in vivo models. 63 In another study, gene shis in TAMs towards M1 type and also induced apoptosis in cancer associated broblasts were observed upon treating with a biodegradable nanoparticle called ONP-302 during in vivo analysis. These particles are negatively charged and were developed from poly(lactic-co-glycolic acid) (PLGA). 64

Liposomes
Made from cholesterol and other natural or synthetic phospholipids, 65 liposomes are one of the most commercially successful nano-drug delivery platforms. 66 Both oral delivery and injection methods are applicable in the case of liposomebased drug delivery systems. 67 FDA-approved intravenous injection is a primary route of administration for various drugs. Besides, subcutaneous, intradermal, intraperitoneal, and intramuscular administrations are also available. The mechanism of liposome drug delivery action relies on the accumulation, uptake, and release of drugs. 68 The interactions with drug and tumor sites happen via passive and active targeting. 69 Passive targeting mostly uses the enhanced permeability and retention effect observed in tumor microenvironments to improve the retention of liposomes at the tumor sites. 70 However, active targeting by surface functionalization has improved the drug-targeting ability of liposomes.
Active targeting of liposomes for drug delivery to tumor sites uses surface functionalization of the lipids surface using various methods. The use of antibodies, small molecules, peptides, or carbohydrates has been tested for surface functionalization. 71 Antibodies, with their specic targeting properties, can be used for targeting cancer-specic surface antigens such as Melanoma cell adhesion molecule (MCAM), 72 Her 2 receptor, CD44, and growth factor receptors such as VEGFR and EGFR. 71 Surface functionalization using small molecules such as folate, estrone, and anisamide are also effective in targeting tumor surface receptors for these molecules. Carbohydrates and proteins such as mannose and Beta FGF which can bind to specic cell surface receptors have also been investigated as potential surface active molecules.
Along with surface molecule based targeting, another method used to target liposomes is the stimuli-sensitive cleavage of coating polymers used to reduce the phagocytotic clearance of liposomes. Polymers such as PEG used for coating liposomes increase their circulation and may have reduced targeting properties. To improve the cancer site targeting by these coated liposomes, various stimuli based cleaving have been used. These methods also use the tumor microenvironment properties such as altered pH, redox potential, and secreted enzymes to induce cleavage of the surface coating, thereby inducing preferential drug release at the tumor site. 71

Extracellular vesicles
Extracellular Vesicles (EVs), released by prokaryotic and eukaryotic cells into the extracellular environment, have recently gained prominence as cancer nano-drug vehicles. EVs may be actively produced by cells in a constitutive or inducible manner, and are naturally nano-scale to micron-sized membrane vesicles encased by phospholipid bilayers. 73 EVs originate from the endosomal compartment. Their biochemical content consists of lipids, proteins, and microRNA, making them a promising candidate for targeted therapy.
EVs have favorable cellular uptake properties due to their biochemical composition and also can be targeted to tumor sites using surface functionalization. 74,75 The inherent properties of EVs being able to cross the blood-brain barrier also make them promising drug delivery platforms for brain tumors. Also, engineered EV shows increased pharmacokinetic ability, drug load stability, and targeted therapy; for example, paclitaxel shows potent anticancer effects in a model of Murine Lewis lung carcinoma pulmonary metastases, and also accumulates in cancer cells upon systematic administration, thereby, improving therapeutic outcomes. 76 However, the non-scalability of EV production is a major challenge to be overcome before it can become an attractive commercial nano-drug vehicle.

Nanoemulsions
Nanoemulsions are water-in-oil or oil-in-water emulsions of nano-dimensions that can be used for drug delivery to tumor sites. 77 With the usage of generally recognized as safe (GRAS) oils as vehicles, nanoemulsions have the advantage of reduced side effects. 78 Similar to liposomes, nanoemulsions also can utilize the EPR effect to passively target the tumor site. However, recently surface functionalization of the outer oil portion of the emulsions are being studied to improve the drug targeting to the specic tumor. 79 Active targeting methods for nanoemulsions also use peptides such as RGD peptide and transferrin, small molecules such as biotin and folate, or certain antibodies specic to tumor surface antigens. 80 Novel targets such as lysophosphatidic acid receptor is also being studied to improve the drug targeting to specic tumors. 79 Advantages of nanoemulsions in their versatility for the drug load promoting combinatorial drug use, inherent ability to overcome multidrug resistance, and the potential ability to use the drug itself as the emulsion without the need for a carrier make them interesting choices for cancer therapy.

Dendrimers
Dendrimers are articial nanocarriers made of radially symmetric arrangements of monomers 81 with a tree or branchlike appearance. 82 They have high surface functionalization and targeting properties owing to their versatility of design. As they can be designed specically to suit each target, dendrimers are one of the nanocarriers with the easiest surface functionalization properties. The ability to precisely regulate the size of the dendrimers also makes it possible for them to be passively targeted to tumor sites via EPR. Dendrimers also can be hybridized into other types of nanocarriers, such as encapsulating them in other polymer shells 83 making it a versatile drug delivery platform for cancer therapy.
Dendrimers have high carrier capacity for the drugs 84 and can be specically engineered for various drug release mechanisms. Some of the commonly used ones include variations in the number of terminal groups and the use of degradable spacers and pH-sensitive linkages. 85 The surface functionalization methods using vitamins, antibodies, peptides, etc., have also been able to overcome the inherent drawbacks of dendrimer-mediated drug delivery such as rapid systemic elimination.

Nanoparticles from inorganic materials
Nanoparticles from inorganic materials are the other kind of drug delivery system widely used for cancer treatment. The commonly used materials for these nanoparticles include gold and silver nanoparticles, carbon quantum dots, carbon nanotubes, metal oxide particles, and mesoporous silica nanoparticles. They are well-known for their precise targeting, good pharmacokinetics prole, encapsulation of poorly soluble drugs, and diagnostic applications. The size and characteristics of nanoparticles are very important when designing drug delivery systems. 86 Normally, nanoparticles act as a carrier system to incorporate active ingredients, and they release the active compound into the targeted site to induce cell death.
3.5.1. Gold/silver nanoparticles. Nanoparticles of gold and silver are capable of delivering small as well as large drug molecules to the tumor site. The major mode of drug targeting involves the use of EPR as well as tumor micro-environmental properties such as altered redox potential and pH. 87 Being metallic, they can also be used for hyperthermic treatment using an external heat source such as microwave, post targeting to the tumor site. However, aggregation and higher cytotoxicity to healthy cells by these metal particles reduce their use unless hybridized with certain polymers such as PEG. 88 3.5.2. Magnetic nanoparticles. Magnetic nanoparticles are used for cancer therapy, especially for hyperthermic treatment including metallic, or metallic shells and oxides. Usually, a magnetic nanoparticle is prepared from pure metal or in combination with polymers. Delivery of magnetic nanoparticles into the targeted site via injection using catheters or hypodermic needles has been demonstrated. 89 A heat source such as a microwave can be used to induce hyperthermia at the specic target, thereby locally increasing the temperature to around 42°C . Higher temperature leads to the death of cancerous cells due to their leaky vascular nature, without a signicant effect on the normal cells. Apart from hyperthermia, magnetic nanoparticles have been used for theranostic, photodynamic therapy, photothermal ablation therapy, biosensors, drug delivery, and MRI imaging 90 because of the super-paramagnetic feature of iron oxide nanoparticles. 91 3.5.3. Carbon quantum dots. Carbon quantum dots (CDQs) are uorescent carbon nanoparticles with imaging and drug delivery applications in cancer. 92 They have high targeting capabilities due to their ease of surface functionalization, and therefore, can be used with multiple targeting molecules on the surface. The uorescence properties of CDQs render them highly capable of bio-imaging, adding to their overall theranostic value. 93 The low toxicity, high biocompatibility, ease of targeting, and added diagnostic functionalities of CDQs make them one of the most promising emerging nano-drug carrier platforms.

Solid lipid nanoparticles (SLN)
SLNs are a colloidal drug delivery system in which the liquid lipid is replaced by solid lipid. 94 Generally, as the name indicates, solid lipid nanoparticles consist of a solid lipid, emulsi-er, and water. Fatty acids, steroids, waxes, triglycerides, and partial glycerides are commonly used as lipids. SLN can be administered through various routes such as parenteral, oral, rectal, ophthalmic, and topical. Solid solution and core-shell models are the two types of SLNs models. 95 It is observed that SLNs have higher drug release due to their high surface area, and the homogeneous distribution of the drugs will lead to slow release. In addition to this, the high mobility of the drug and crystallization behavior of lipid carriers also facilitates faster release. 96 SLN is delivered into the targeted area by passive, active, and co-delivery mechanisms. Passive targeting is mainly achieved due to EPR effects, and active targeting is by recognition of receptors or transporters over-expressed on the surface of tumor cells. The co-delivery method is applied by delivering two compounds to the drug delivery system. 97 Enhanced pharmacokinetics and efficacy against multidrug-resistant cancer cells were observed in cancer cell lines such as MCF-7, A549, and MDA-MB-231 when treated with vorinostat-loaded solid lipid nanoparticles. 98

Status of approved drugs and those under clinical trials
Clinical trials are the last stage of drug development wherein the drug formulations are tested on humans to determine their actual efficacy and side effects, to obtain approval for commercial use of the drug formulation. 99 There are various phases for the clinical trial of a drug and all of them have to be cleared sequentially for the drug to be approved for medical use against the disease. The duration of each phase, the conditions involved, and the number of people the drug is tested on at each phase is decided by the drug regulatory authority. However, there mostly involves four phases of clinical trials before a drug is approved for medical use.
Phase I of a clinical trial involves less than a hundred people and may include healthy people as control groups as this phase is to determine the safety and dosage of the drug. However, for cancer related drug trials, it is mandatory that the group includes people with that particular type of cancer. 99 Aer clearing phase I, the drug can enter phase II clinical trials which is conducted on a few hundred people with a particular cancer. The objective of this phase of the clinical trials is to determine the efficacy and side effects. Therefore, it is common to have double blind studies with placebo control groups for this phase. The next phase also is aimed at determining the side effects. However, the focus is on long term and less common side effects and therefore include a larger study group of up to a few thousand people and can go on up to 3-4 years. Upon successful completion of phase III clinical trials, the drug formulation can be approved and may be marketed for medical use. However, the monitoring continues as phase IV wherein any and all adverse reactions reported are investigated for determining the overall safety and efficacy of the drug.

Approved nano-formulations for cancer therapy
Nano-formulations have been marketed for medical use against cancer since early 90's with the polymer-protein conjugate Zinostatin stimalamer 100 being approved in Japan against hepatocellular carcinoma and the pegylated liposome Doxil® which was marketed as an anti-ovarian cancer drug formulation in the United States of America. 101 With time, many other types of nano-formulations including liposomes, metal and metal oxide nanoparticles, polymeric micelles, and lipid nanoparticles have been developed and cleared for medical use by multiple agencies all over the world, with many more under various stages of clinical and preclinical trials. Table 1 gives a list of nano-formulations and the drugs used in them, which have already been approved for medical use.
Doxil® was the rst liposome to get approval in the US in 1995 101 for the treatment of ovarian cancer and AIDS-related Kaposi's sarcoma. Aer a year, NeXstar Pharmaceuticals developed daunorubicin-loaded NPs (DaunoXome®) to treat HIVassociated Kaposi sarcoma. In 2000, Myocet® is another formulation that contains doxorubicin and cyclophosphamide and got EMEA approval for treating metastatic cancer. Later, Marqibo® got FDA approval for treating non-Hodgkin's lymphoma and leukemia. In 2013, Lipusu was developed by incorporating paclitaxel for treating gastric, ovarian, and lung cancers. 101 It is understood that the addition of other compounds such as cholesterol and PEG will help attain the desired properties. 102 Mifamurtide-loaded liposomes were developed by Takeda Pharmaceutical Limited to treat highgrade non-metastatic osteosarcoma. In 2017, Vyxeos got FDA approval. It is a liposomal formulation of daunorubicin and cytarabine. The investigators experimented on 309 patients with an average age range between 60-75 to treat acute myeloid leukemia (t-AML) or acute myeloid leukemia (AML) with myelodysplasia-related changes (AML-MRC). 103 Along with it, Irinotecan-loaded PEGylated liposome (Onivyde) and cytarabine-loaded liposome (DepoCyt) also got approval for pancreatic adenocarcinoma and lymphomatous meningitis treatment respectively. 104 Compared to liposomal preparations, other types of approved nano-formulations are lesser in number. However, the focus on the other types of nano-formulations have also been present and a few are approved for clinical use. Styrene maleic anhydride neocarzinostatin (SMANCS) loaded polymer protein conjugate got approval in Japan (1994) for treating renal carcinoma. 105 Eligard® is a leuprolide acetate-loaded polymeric nanoparticle that got FDA approval in 2002 for prostate cancer. 106 Another formulation is nanoxel® composed of Nisopropyl acrylamide and vinylpyrrolidone monomers and loaded with Doxetaxel. It got approval in India for the treatment of metastatic breast cancer, ovarian cancer, NSCLC, and AIDSrelated Kaposi's sarcoma. 107 Apealea is another drug that got approval from EMA for treating epithelial ovarian cancer, primary peritoneal cancer, and Fallopian tube cancer. It is a paclitaxel-containing polymeric micellar formulation. 108 Ferucarbotran (carboxydextran coated) and Ferumoxide (dextran) coated were two iron oxide nanoparticles that got approval for cell labeling, especially in the USA. 109 NanoTherm is an EMAapproved drug for treating glioblastoma, prostate, and pancreatic cancers. It is a nanoparticle of superparamagnetic iron oxide coated with aminosilane. Moderate adverse effects were identied with NanoTherm and it is good to increase blood circulation time and tumor uptake. 110

Nano-formulations under clinical trials
The number of nano-formulations under clinical trials has been increasing in recent years with many trials in phase II and III at present. Even though liposomes and its hybrids still dominate the trials, there are many metal oxide nanoparticles, carbon quantum dots, polymeric micelles, nanoemulsions and other types of nano-formulations have also been gaining interest ( Table 2). Several phase I trials using nano-platforms for use as drugs, as well as a combined theranostic platforms have reported positive results. Many of the nano-formulations under trial are for developing effective nanocarriers for established and approved anticancer drugs. More than one study is in phase I trial to test the efficacy of Topotecan-based liposomes for advanced solid tumors (NCT04047251), 99 small cell lung carcinomas as well as ovarian carcinomas (NCT00765973). 111 Even though the phase I trials have been completed, no results have been published. Another topoisomerase inhibitor undergoing clinical trials for a liposome mediated delivery is Irinotecan (NCT04796948). 112 This study looks into the safety and tolerability of Irinotecan in combination with 5FU and oxaliplatin and is in the initial stages of phase I trials. Paclitaxel (NCT00080418) 113 and Doxetaxel (NCT01151384) 114  doxorubicin PEGylated liposomes (NCT03591276) 122 and lipid nanoparticles (NCT05267899). 123 Another emerging eld with many ongoing phase I clinical trials are nanomedicine platforms for imaging as well as for radiation therapy. CdS/ZnS core-shell type quantum dots with carboxylic acidfunctionalized (QDs-COOH) are being studied for their bioimaging capabilities as uorescent nanoparticles conjugated somatostatin analogue (NCT04138342). 124 Other metal/metal oxide nanoparticles studies include FerumoxytolIron oxide nanoparticles for hepatic cancers (NCT04682847), 125 CD24 primer and gold nanoparticle being investigated as a diagnostic tool and biomarker (NCT04907422) 126 for salivary gland tumors, Ferumoxtran-10 Superparamagnetic nanoparticle (NCT00147238) 127 for detection of cancer in the pelvic lymph nodes or malignant pelvic lymph nodes and Hafnium Oxide Particle activation by radiation therapy against locally advanced or borderline-resectable pancreatic cancer (NCT04484909). 128 Other studies with antibody based drugs, miRNAs, and targeted therapy against gene targets such as DOTAP: Chol- Recently, in phase II clinical trial (NCT02596373) 131 mitoxantrone hydrochloride liposome (Lipo-MIT) was tested in patients with advanced breast cancer. Lipo-MIT was injected intravenously and the efficacy and safety in terms of objective response rate, disease control rate, and progression-free survival were investigated. An altered toxicity prole was observed with Lipo-MIT treatment with fewer cardiovascular events. But this study was limited due to its small sample size. 132 In another study, the bioequivalence of a hybrid pegylated liposomal doxorubicin (PLD) hydrochloride injection with reference product Caelyx® was evident in patients with ovarian cancer. 133 Another study reported promising efficacy and fewer toxic effects in platinum-resistant recurrent ovarian cancer patients treated with a formulation containing apatinib and pegylated liposomal doxorubicin. This was a comparative study conducted with and without the addition of apatinib in pegylated liposomal doxorubicin. However, some side effects such as hypertension and decreased neutrophil and white blood cell count were observed in patients who underwent this combinatorial therapy. 134 Recently, a phase 1 study of Eribulin liposomal formulation (E7389-LF) was examined for breast cancer treatment. Patients received formulation every three weeks and tumor assessment was conducted once in six weeks. Tolerability and antitumor activities were evident in the investigation. However, adverse events such as neutropenia, leukopenia, and thrombocytopenia were observed in patients. 135 Good efficacy of combinational drugs containing pembrolizumab, bevacizumab, and pegylated liposomal doxorubicin was observed in ovarian cancer patients. But four patients had palmar-plantar erythrodysesthesia. 136 Liposomal Gemcitabine (FF-10832) is a stable liposome that was tested for antitumor activity in advanced solid tumor patients. Better efficacy and fewer adverse  ects were reported in this study. 137 ThermoDox® is a thermally sensitive liposome loaded with doxorubicin. This particle is triggered by mild hyperthermia and the quantication of accumulated drug concentration can be examined before and aer ultrasound exposure. Results showed that this formulation is better for drug delivery and cell penetration. 138 The percentage of studies which clear the phase II clinical trials to reach the phase III clinical trials are much lesser as compared to those which reach phase II form phase I. The major reason for this is the higher number of participants, thereby increasing the probability of determining the side effects. 99 There have been a few studies that had to be dropped at phase II and III due to non-availability of participants as well. Phase III clinical trials have even more stringent requirements and are fewer in number. There are two carbon nanoparticlebased cancer nanomedicines currently under phase III trials (NCT04759820), 139 NCT02123407). 140 A paclitaxel micellar nanoparticle (NCT01644890) 141 and a Doxorubicin liposome formulation (NCT05561036) 142 are also undergoing phase III trials. Some of the formulations have also reached phase IV and have been approved for marketing (NCT00606515), 143 NCT03817515). 144 The numbers of effective and safe nanoformulations clearing clinical trials are still very few and there are still challenges ahead to be cleared before cancer nanomedicine can be utilized to its full potential.

Conclusion and future challenges
In the past years, nanotechnology has been widely applied in all aspects of science, engineering, and technology, and research & development in this discipline has been intense. Many cancer nanomedicine researchers have investigated largely consistent processes, which include formulation, characterization, in vitro proof of concept, and validation of anticancer activity in preclinical trials. Low throughput and weak predictive abilities plague validation efforts. This review has covered the evolution of cancer nanomedicine from its inception to its current stateof-the-art. In the beginning, we explained the concept of "nanotechnology" and its advancement during the next years, especially in the case of cancer nanomedicine. From 1959 to till now, advancement in nanotechnology is visible in each and every eld, including clinical areas. Some of the formulations got approval and many in vitro, in vivo, and clinical studies are ongoing. Increased circulation time, suitable size range, less toxicity, and good drug accumulation made it superior to the conventional method. Nanoparticles can target a cancer cell via passive and active targeting. Due to EPR effect and small size, nanoparticles can easily enter into the tumor cells and retain them in the tumor bed due to long circulation time. Besides, targeting TME is also the best way to reduce tumor progression. Different types of nano-formulations such as liposomes, extracellular vesicles, nanoemulsions, gold/silver nanoparticles, magnetic nanoparticles, carbon dots, and solid lipid nanoparticles are investigated for tumor targeting, bio-imaging, and drug delivery. However, new methods/or technologies are always under consideration to overcome the current challenges explained below. To develop nano-therapeutics of high efficacy with accumulation in the tumor site rather than the normal cells should be the main criteria focused by the researchers. Because of the composite structure of the nanoparticles, it is very difficult to identify their toxicity and there is no proper validated model for checking the nano-bio interactions, especially, nano-immuno interactions. Other two factors hindering the approval are reproducibility and transparency. Apart from that, the higher cost and complexity have added restriction to the translation success. The journey of a drug from its manufacturing stage to the approval stage is very complex and time-consuming. In addition, the advancement in the instrumentation and characterization methods are necessary for the accomplishment of a drug product. 145 Moreover, choice on the drug selection with a combinatorial regimen for the specic disease over the targeted population should also be considered important. Most of the time biological barriers also cause an imbalance in terms of targeted delivery, permeability and penetration, endo/lysosomal escape, intracellular processing and trafficking, and metastasis. 146 The lab-scale production of drugs are easier to achieve, but large-scale manufacturing seems to be a challenging task due to limitations in the advanced experimental set-up and nonavailability of sufficient information on the scale-up technologies. Also, adverse effects are encountered due to the difficulties observed during the scale-up process and in reproducing the preparation process. 147 The developed nanomedicines, are in general, screened by FDA and EMA, but the lack of standard operating procedure for the evaluation of nanomedicines makes the task tougher. 148 Checking the drugs for any changes during every phase of the clinical trials seems crucial in terms of safety and biocompatibility. Moreover, well-dened characteristics and reproducibility of the drug are essential for initiating the clinical trials. Challenges faced during nanomedicine manufacturing include the presence of contaminants, poor quality control, insufficient batch-to-batch variability, chemical instability, biocompatibility, low production yield, scalability complexities, high cost, lack of infrastructure, government regulations and lack of funding. 145,149 Patient stratication, drug selection, combination therapies and immunomodulation are some of the important parameters to overcome the challenges of nanoparticles 150 and these details are illustrated in Fig. 5. Various approaches like CTC analysis, immunohistochemical assessment, and imaging of accumulation of nanomedicines are used for patient stratication. 151,152 Liquid biomarkers, tissue biomarkers, and imaging biomarkers are currently available. While discussing drug selection, drug classes' modular design and library screening could be considered. Nowadays, combination therapies are widely used in cancer research to increase the efficacy. However, clinical trials for nanomedicines are mostly designed to evaluate monotherapy, which would affect the progress in clinical trials. Improving cancer therapy with the capability to target and modulate the component of the immune system is needed. 146 A well-designed nanoparticle can be applied as a potential tool for bio-imaging and tumor detection. The high sensitivity of the nano-biosensors is due to the high surface area to volume ratio of the nanoparticle, most preferentially, the gold nanoparticles, quantum dots and polymer dots. 153 Also, radio-labelled nanoparticles, uniquely suited for positron-emission tomography (PET) or single-photon emission computed tomography (SPECT) mapping of sentinel lymph node and T2-weighted magnetic resonance imaging (MRI) probes are mostly made from iron oxide nanoparticles. 154 To improve the drug delivery, we have to consider every step ranging from internalization, circulation, penetration into the tumor microenvironment, binding to the target and the destruction of tumor cells. It is necessary to have a clear understanding of the relationship between the physicochemical properties of nanoparticles and their biological response. In recent years, researchers are also focusing on computer algorithms for achieving proper drug delivery. 145 Opportunities and challenges are two sides of a coin. More clinical trials are needed for the conrmation of preclinical and in vitro studies. These should help develop a deep understanding of the interaction of nanoparticles with cells and its aer-effects. The main drug delivery routes are oral, intravenous, and subcutaneous for anticancer administration. Inhalation delivery, rectal delivery, and pulmonary delivery are newly proposed. However, these methods are limited due to the high toxicity caused by the combined action of drug deposition and high therapeutic potency. Besides, massive drug doses are needed due to the loss of drugs in the pulmonary tract. In rectal delivery, low absorption is also a major problem. 155 Recently, mitochondria-based targeting has shown great promise in cancer therapy due to the involvement of mitochondria in tumor generation and progression. Difficulties in clinical trials and bio-safety concerns due to the positive charge on nanoparticles (toxic to normal cells) are the major challenges faced during mitochondrial-targeted therapy. 156 Another study mentioned that liposomal formulations lose their activity overtime due to their exposure to blood proteins. 157 The major drawback of carbon-based nanomaterials is the difficulty in standardization due to their non-uniform nature; the phototherapy method is associated with a lack of targeting. Besides, some tumors are difficult to identify due to their small size. Therefore, diagnosis techniques must be improved. 158 Surely, all the challenges associated with cancer nanomedicine therapy will eventually disappear and, in the future, it will play a crucial role in all aspects of cancer therapy including diagnosis, bio-imaging, and drug delivery.

Conflicts of interest
The authors report no conicts of interest in this work.